Work in our laboratory has demonstrated that repeated stresses and withdrawals leads to sensitization of the withdrawal-induced anxiety to a brief exposure to chronic ethanol, in further support of the Ballenger and Post (1978)"kindling" hypothesis. A major finding is that flumazenil, a benzodiazepine (BZD) antagonist, minimizes the sensitization of anxiety following the repeated withdrawal protocol. Based upon these data with flumazenil, it is presumed that an endogenous substance having a negative effect on GABAA receptors is responsible for the sensitization of anxiety induced by the repeated stress sensitization of anxiety. In support of this hypothesis, two doses of DMCM, a BZD-inverse agonist, given instead of withdrawal results in sensitization of anxiety during a final withdrawal. The purpose of the present investigation is to test specific hypotheses to account for these findings. Specific Aim 1 will determine whether repeated stress sensitization of withdrawal-induced anxiety will be antagonized by flumazenil and whether this treatment results in an increase in mRNA expression for diazepam binding inhibitor (DBI), an endogenous BZD-inverse agonist, allowing identification of brain sites where adaptive change in DBI could be contributing to withdrawal-induced anxiety. Specific Aim 2 will test whether the repeated stresses or exposure to a BZD-inverse agonist will persist in inducing sensitization of withdrawal-induce anxiety upon later re-exposure to chronic ethanol, as occurs with repeated withdrawals. Additionally, we will determine if exposure to stress or a BZD-inverse agonist during multiple withdrawals will extend the duration during which later re-exposure to chronic ethanol will continue result in sensitization of anxiety. Specific Aim 3 will determine if microinjection of flumazenil into brain regions identified in Specific Aim 1 will block sensitization of withdrawal-induced anxiety following multiple stresses and whether microinjection of a BZD-inverse agonist into the selected sites will sensitize withdrawal-induced anxiety. Specific Aim 4 will test whether GABA(A) and BZD binding is affected by repeated stresses and a single withdrawal and whether responsiveness to a BZD-inverse agonist is increased by the repeated stresses. This will be complemented by experiments to identify whether an endogenous compound sensitive to flumazenil increases with repeated withdrawals. Defining the basis of the pathological adaptive mechanism(s) responsible for "kindling" of withdrawal-induced anxiety associated with repeated stresses may provide greater insight into alcohol abuse and provide new avenues for treatment.